D-allose is an uncommon sugar that rarely has been detected in nature. It has been reported to be present in plant glycosides (Guido W. Perold and Peter Beylis, "Metabolites of Proteaceae," VIII Nature, Rubropilosin and Pilorut, 1973, J. Chem. Soc., Perkin Trans. 1, (6), 643-9 and G. W. Perold; P. Beylis and A. S. Howard, "Occurrence of D (+)-allose in Nature", 1971, J. Chem. Soc. D., (11), 597) and in the external polysaccharides of a bacterium (Akira Misaki, Yoichi Tsumuraya and Mariko Kakuta, "D-Allose-Containing Polysaccharide from Methanol by Pseudomonas Sp.", 1979, Carbohydrate Research, 75, C8-C10 and Toyo-Soda, "Production of an Allose-Containing Polysaccharide by Cultivation of Pseudomonas Viscogena," 1984, U.S. Pat. No. 4,425,431.) It has not been reported to exist free in solution in nature. A bacterium previously has been described that can utilize D-allose as a sole source of carbon and energy for growth. This microbe was found to contain an ATP linked D-allose kinase enzyme activity (L. N. Gibbins and F. J. Simpson, 1963, Can. J. Microbiol, 9, 769 and R. P. Mortlock, "Catabolism of Unnatural Carbohydrates by Microorganisms," VIII, D-allose, 1976, Advances Microbial Physiology, V. 13, 43-45.) The product D-allose-6-phosphate could be metabolized further by isomerization to a ketose sugar.
D-allose can be synthesized by chemical epimerization of D-glucose. (Blaise J. Arena, Raymond J. Swedo and Bruce E. Firth, "Molybdate-Containing Anion Exchange Resins for Continuous Interconversion of Epimeric Aldoses and Aldose Analogs,"1988, U.S. 88-288375). This epimerization is not specific and is accompanied by formation of D-mannose and D-altrose. Some pentose sugars structurally similar to D-ribose also are formed. The unique structure of D-allose gives it certain special properties; U.S. Pat. No. 4,963,382. D-allose is not readily metabolized by animals and humans and is easily excreted. Although it is very similar to D-glucose in its taste and physical properties it is non-caloric.
In recent years the role of carbohydrates in cell recognition has become established; N. Sharon and H. Lis, Scientific American, January, 1993, pp 82-89. With this has come an increasing realization of the potential importance of carbohydrates, either per se or as a discrete portion of a more complex molecule, as pharmacologically active agents. See, for example, Genetic Engineering News, July, 1994, p. 6 et if. An indispensable prerequisite of pharmaceutical testing, of in vivo studies generally, and of manufacturing processes is an accurate, quantitative, highly specific analytical procedure for D-allose, especially in the presence of normal bodily fluids and in the presence of similar carbohydrates, both pentoses and hexoses. This application is directed to just such an analytical procedure.
In particular, we have isolated a fungus which elaborates a dehydrogenase specific to D-allose. Utilization of the dehydrogenase with nicotinamide adenine dinucleotide (NAD) as a cofactor enables the development of both a simple qualitative test for the presence of D-allose as well as a more elaborate quantitative analysis for D-allose which remains specific to the substrate even in the presence of other materials one could expect to be present under typical analytical conditions.